The present invention relates to methods and kits for directed differentiation of embryonic stem cells and their use in vivo and in vitro.
Embryonic stem (ES) cells have been described in the prior art as undifferentiated pluripotent cells derived from the inner cell mass of blastocyst stage embryos which can grow indefinitely in culture while retaining a normal karyotype. Blastocyst derived stem cells form teratomas when injected into muscles or testis of severe combined immunodeficiency mice. Murine embryonic stem cells have been reported to form aggregates or embryoid bodies in vitro which are capable of spontaneously differentiating into various cell types. In contrast to blastocyst derived stem cells, embryoid body derived cells do not appear to form teratomas when implanted in vivo. (Robertson, 1987; Thomson et al, 1998; Reubinoff et al, 2000).
While human embryonic stem cells have been recovered from human embryos produced by in vitro fertilization, the formation of embryoid bodies from non-human primates and from humans has been problematic. In contrast to mice, the formation of embryoid bodies from primates, more particularly, the common marmoset (Thomson et al, 1996; U.S. Pat. No. 5,843,780) and the rhesus monkey (Thomson, 1995) were found to be inconsistent and asynchronous. Moreover, differentiation of rhesus embryonic stem cells was disorganized and vesicular structures did not form. In addition, human embryonic stem cells have been shown to differentiate in a spontaneous and uncontrolled manner, so that the experimenter cannot determine which cell types will form in vivo or in vitro (Thomson et al, 1998; Reubinoff et al, 2000; Itskovitz-Eldor et al 2000).
Development of techniques for manipulating differentiation of human embryonic stem cells to provide a uniform population of precursors and differentiated cells of a desired lineage are desirable for in vivo medical uses and for in vitro assays.
Although many useful lessons may be learned about development in mammals from studying mice, not all of development is the same in all animals. Consequently, it is desirable to have tools to analyze and compare pathways in different mammals and to combine these tools with a methodology that permits the isolation, preservation and cultivation of embryonic stem cells from mammals including humans for correcting a disease condition resulting from cell degeneration.
In particular, human embryonic stem cells may be uniquely useful as a source of cells for transplantation in numerous human pathologies, and as a component in biomedical engineering as well as providing clues on early stages of human development.